Abstract
.SignificanceLight-field microscopy (LFM) enables fast, light-efficient, volumetric imaging of neuronal activity with calcium indicators. Calcium transients differ in temporal signal-to-noise ratio (tSNR) and spatial confinement when extracted from volumes reconstructed by different algorithms.AimWe evaluated the capabilities and limitations of two light-field reconstruction algorithms for calcium fluorescence imaging.ApproachWe acquired light-field image series from neurons either bulk-labeled or filled intracellularly with the red-emitting calcium dye CaSiR-1 in acute mouse brain slices. We compared the tSNR and spatial confinement of calcium signals extracted from volumes reconstructed with synthetic refocusing and Richardson–Lucy three-dimensional deconvolution with and without total variation regularization.ResultsBoth synthetic refocusing and Richardson–Lucy deconvolution resolved calcium signals from single cells and neuronal dendrites in three dimensions. Increasing deconvolution iteration number improved spatial confinement but reduced tSNR compared with synthetic refocusing. Volumetric light-field imaging did not decrease calcium signal tSNR compared with interleaved, widefield image series acquired in matched planes.ConclusionsLFM enables high-volume rate, volumetric imaging of calcium transients in single cell somata (bulk-labeled) and dendrites (intracellularly loaded). The trade-offs identified for tSNR, spatial confinement, and computational cost indicate which of synthetic refocusing or deconvolution can better realize the scientific requirements of future LFM calcium imaging applications.
Highlights
Understanding how neuronal networks learn, process, and store information requires imaging techniques capable of monitoring the activity of hundreds to thousands of neurons simultaneously in three-dimensional (3D) tissues
We show that Light-field microscopy (LFM) can resolve calcium transients simultaneously in axially separated somata and dendrites of neurons loaded with a red-emitting calcium dye, CaSiR-1.23 We examined trade-offs between the tSNR and the spatial signal confinement of calcium signals localized in volumes reconstructed from light fields by synthetic refocusing and 3D deconvolution
The tSNR, peak signal, and baseline noise were compared between the two light-field reconstruction algorithms and widefield image series
Summary
Understanding how neuronal networks learn, process, and store information requires imaging techniques capable of monitoring the activity of hundreds to thousands of neurons simultaneously in three-dimensional (3D) tissues. Synthetic and genetically encoded fluorescent indicators of intracellular calcium concentration[2,3] and membrane voltage[4,5] enables functional imaging on these scales. Scanning limits the fluorescence bandwidth and the acquisition speed and temporal signal-to-noise ratio (tSNR). That is why applications requiring high acquisition rates and/or SNR typically rely on widefield, single-photon imaging to maximize photon flux by exciting fluorescence simultaneously in all illuminated structures. Widefield excites fluorescence efficiently throughout a volume, only one axial plane is in focus. In this configuration, fluorescence excited above and below the imaging plane is unnecessary and contributes spurious fluorescence to the in-focus image, degrading contrast and confusing the functional signals.[6]
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